Adverse drug metabolism, drug-drug interactions, and toxicity effects cause numerous drug failures. High throughput screening (HTS) technologies to predict drug toxicity based on chemical structures have the potential to reduce pipeline attrition. Reaction Biology Corporation has developed extremely low cost nanoliter reaction microarrays to serve markets for HTS in drug discovery, large scale IC50 determinations, and selectivity profiling. These reactions are 10,000-fold smaller than well plate formats currently used in drug discovery. Microarrays allow hundreds antigens to be measured in 1000's of lysates obtained from cells treated with compounds. Specific aims in this Phase I are: Aim 1: Lysis and Printing. Design and implement microarray surface chemistry for optimized antibody-coating uniformity and stability. Develop robust high throughput cellular lysis protocols for efficient antigen release and recovery. Lysis and printing protocols will be developed for up to 6600 lysates printed per microarray. Aim 2: Detection. Optimize and calibrate detection sensitivity down to the 10 pg/ml level for each antigen of interest. Three types of detection will be tested, including: (1) direct fluorescent secondary antibody detection, (2) precipitated enzyme products, and (3) soluble fluorogenic enzyme substrates. This aim will measure HSP70 in HepG2 lysates by the three detection methods. Aim 3: Five-antigen Panel. Establish, calibrate, and validate 5 toxicity biomarkers using the lysis methods developed in Aim 1 and detection methods of Aim 2. Aim 4: Drug-drug interaction. Screen several test compounds for interactions with a pharmaceutical library in HepG2 and measure the five biomarkers developed in Aim 3. We seek to create a database that includes the chemical information of the drug candidates and the protein expression information generated from Aim 4 to establish structure-toxicity relationships (STR). In Phase II, RBC will expand this technology to an extensive panel of protein toxicity biomarkers and a full common drug library.